Electrochemical approach for cancer detection

ABSTRACT

An electrical probe configured to measure an electrical response from a biological cell includes a microwire having a sharpened tip, a catalyst layer formed on the sharpened tip of the microwire, and an array of nanotube electrodes vertically aligned on the catalyst layer.

CROSS REFERRENCE TO RELATED APPLICATION

The present application claims priority from pending U.S. ProvisionalPatent Application Ser. No.62/263,616, filed Dec. 5, 2015, entitled “ASINW-ECIS BIOSENSOR”, which is incorporated by reference herein in itsentirety.

TECHNICAL FIELD

The present disclosure generally relates to a tungsten (W) supportedsilicon nanotube (SiNT) based electrical probe (designated hereinafteras “SiNT/W probe”), a method for fabrication thereof, and applicationsthereof in detecting cancerous state of a single cell.

BACKGROUND

Cancer is recognized as a type of disease that affects many biochemical,electrical, and mechanical functions of a cell. Cytoskeletalalterations, damping of electrodynamic microtubule oscillations,diminution of dielectric properties of the membrane, and disruption inthe ion channel activity are some of the considerable mechanical andelectrical alterations in cells during cancerous transformation.

Highly accurate methods for monitoring such alterations in single cells,such as electrical, mechanical, and electro-optical monitoring of singlecells may detect cancerous transformation in its early stages. In caseof electrical recording, high spatial resolution contacts betweenelectrical probes and single cells and also non-invasive recording arecritical for both fundamental biophysical studies and diseasemonitoring; particularly for bioelectrical signals, which are weakerthan action potentials.

Nanoscale electrical probes (e.g., conductive silicon nanowires andsilicon nanotubes (SiNT)) have opened new fields of investigation,leading to the emergence of possible future applications in cellbioelectrical and electrophysiological studies. Recently, thenanostructured probe-based electrical recording methods have beenapplied solely for action potential measurements outside the cell ofsome special types of electrically active cells with sharp responses,such as neurons and cardiomyocytes.

Therefore, there is a need for a label-free cancer diagnosis or cancerprogression detection method with single-cell resolution usingnon-invasive devices or instruments capable of measuring intracellularbioelectrical responses, even minor electrical variations for a widerange of cell types.

SUMMARY

In one general aspect of the present disclosure, an electrical probe formeasuring an electrical response from a biological cell is disclosed.The probe includes a microwire having a sharpened tip; a catalyst layerformed on the sharpened tip of the microwire; and an array of nanotubeelectrodes vertically aligned on the catalyst layer. The array ofnanotube electrodes are configured to measure an electrical response ofa biological cell in contact with the electrodes.

The above general aspect may include one or more of the followingfeatures. The microwire may include a tungsten microwire with a diameterless than about 2000 μm having a sharpened tip with a diameter about 200nm. The catalyst layer may include a bilayer having a Nickel layer (Ni)with a thickness of about 10 nm to about 40 nm and a Gold layer (Au)with a thickness of about 1 nm to about 4 nm. The nanotube electrodesmay include a plurality of silicone nanotubes (SiNTs).

In another general aspect of the present disclosure, a method forfabricating a SiNT/W probe is described. The exemplary method mayinclude the steps of sharpening one end of a tungsten (W) microwire toform a tungsten (W) needle having a sharp pointed tip, cleaning thetungsten (W) needle to form a cleaned tungsten (W) needle, forming acatalyst bilayer on the sharp tip of the cleaned tungsten (W) needle,growing a plurality of silicon nanotubes (SiNTs) on the catalyst bilayerto form a SiNT/W needle, transferring the SiNT/W needle into a dopingfurnace to form a doped conductive SiNT/W needle, and coating a goldlayer on top of the SiNTs of the doped conductive SiNT/W needle to forma SiNT/W probe. The electrical probe is configured to measure anelectrical response of a biological cell contacting the siliconnanotubes (SiNTs).

In one exemplary implementation, the one end of a tungsten (W) microwiremay be sharpened through an electrochemical etching process. Thetungsten (W) needle may be cleaned via immersion in a solution, forexample a solution of acetone and buffer HF. The catalyst bilayer on thesharp tip of the cleaned tungsten (W) needle may be formed via atwo-step deposition process using an electron beam coating system. Thetwo-step deposition process may include steps of: holding the cleanedtungsten (W) needle under a gold plume to coat a layer of gold on thesharp tip to form a first catalyst layer and holding the cleanedtungsten (W) needle having the first catalyst layer under a nickel plumeto coat a layer of nickel over the first catalyst layer to form thecatalyst bilayer (Ni—Au) on the sharp tip of the cleaned tungsten (W)needle. The plurality of SiNTs can be grown via a vapor-solid-liquid(VLS) process using a Low-Pressure Chemical Vapor Deposition (LPCVD)system. The doping furnace may include a phosphorous doping furnace. Thegold layer may be coated on top of the SiNTs of the doped conductiveSiNT/W needle by assistance of a sputtering system.

In another general aspect of the present disclosure, a single-cell-basedelectromechanical method for cancerous state detection is described. Theexemplary method includes the steps of: preparing a suspension ofindividually suspended biological cells, extracting a single cell fromthe suspension, holding the extracted single cell from the suspension,measuring a first electrical response of the held single cell,step-wised mechanical aspirating the held single cell to form amechanically deformed cell; and measuring an electrical response of theheld single cell after each step of mechanical aspirating. The cancerousstate of the single cell is determined based on the changes in themeasured electrical responses.

In one exemplary implementation, the biological cells include biologicalcells having an elastic cell membrane.

In some exemplary implementations, the suspension of individuallysuspended biological cells may be prepared through steps of culturing aplurality of biological cells onto a substrate, washing the culturedcells, trypsinizing the cultured cells to detach the cultured cells fromthe substrate and form a solution, and centrifuging the solution toseparate a cell suspension including individually suspended biologicalcells.

In some exemplary implementations, the single cell may be extracted andheld by assistance of an electrically activated micropipette. Theelectrically activated micropipette may include a glass micropipettecoated with an electrically conductive layer, particularly a gold layerhaving a specific thickness of about 10 nm.

In some exemplary implementations, the electrical response of the heldsingle cell may be measured by assistance of an electrical probe. Theelectrical probe includes tungsten-(W—) supported siliconnanotube-(SiNT-) based (SiNT/W) electrical probe.

In another exemplary aspect of the present disclosure, anelectromechanical system for detecting cancerous state of a single cellis described. The exemplary system includes an aspirating mechanism andan electrical measurement mechanism. The aspirating mechanism may beconfigured to extract and hold a single cell and apply a mechanicalaspiration to the single cell, and the electrical measurement mechanismmay be configured to measure an electrical response of the single cell.The cancerous state of the single cell may be detected based on thechanges of the measured electrical responses.

In some exemplary implementations, the aspirating mechanism may includean electrically activated glass micropipette coated with a gold layerand having two ends. The electrically activated glass micropipette maybe assembled on a microinjection microscope from one end and having anozzle at the other end, the nozzle may be configured to apply andtransfer the mechanical aspiration.

In some exemplary implementations, the electrical measurement mechanismmay include an electrical probe, configured to connect to the extractedand held single cell, a signal controlling system, configured to applyan electrical signal to the extracted and held single cell connected tothe electrical probe and to acquire an electrical response correspondingto the electrical signal from the extracted and held single cellconnected to the electrical probe; and a data processor, configured torecord and analyze the electrical response to detect the cancerous stateof the single cell.

In some implementations, the electrical probe may include atungsten-supported silicone nanotube-based (SiNT/W) probe.

In some implementations, the signal controlling system may include an ACsignal source configured for applying the electrical signal to theelectrical probe, and a data acquisition module configured for acquiringthe electrical response corresponding to the electrical signal from theelectrical sensors. In some exemplary implementations, the AC signalsource may apply a voltage of about 40 mV to the electrical sensors.Correspondingly, the applied voltage may have a frequency in a range ofabout 100 Hz to 100 KHz.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of one example of a SiNT/W probe, consistent withone or more exemplary embodiments of the present disclosure.

FIG. 2 illustrates an example method for fabricating a SiNT/W probe,consistent with one or more exemplary embodiments of the presentdisclosure.

FIG. 3 is a schematic of one example of a tungsten (W) microwire, atungsten (W) needle, and a cleaned tungsten (W) needle with a catalystbilayer on the sharp tip and a SiNT/W needle, consistent with one ormore exemplary embodiments of the present disclosure.

FIG. 4 illustrates an example of a single-cell-based electromechanicalmethod for cancerous state detection, consistent with one or moreexemplary embodiments of the present disclosure.

FIG. 5A is a schematic of an example single biological cell held andaspirated by assistance of an electrically activated micropipette andconnected to a SiNT/W probe, consistent with one or more exemplaryembodiments of the present disclosure.

FIG. 5B is a schematic of actin microfilament distribution for anexample of a non-aspirated cell, consistent with one or more exemplaryembodiments of the present disclosure.

FIG. 5C is a schematic of actin microfilament distribution for anexample of an aspirated cell, consistent with one or more exemplaryembodiments of the present disclosure.

FIG. 6 illustrates an example of an electromechanical system fordetecting cancerous state of a single cell, consistent with one or moreexemplary embodiments of the present disclosure.

FIG. 7A illustrates an optical image of an example of a sharpened tip ofa tungsten (W) needle, consistent with one or more exemplary embodimentsof the present disclosure.

FIG. 7B illustrates a field emission scanning electron microscope(FESEM) micrograph of an example of grown array of SiNTs over thesharpened tip of a W needle, consistent with one or more exemplaryembodiments of the present disclosure.

FIG. 7C illustrates a field emission scanning electron microscope(FESEM) micrograph of an example of a single long free end siliconenanotube (SiNT) among an array of SiNTs grown over a sharpened tip of aW needle, consistent with one or more exemplary embodiments of thepresent disclosure.

FIG. 8A illustrates an optical image of an example of a singlebiological cell held by assistance of an electrically activatedmicropipette and a long free end SiNT of a SiNT/W probe placed in acellular media solution, consistent with one or more exemplaryembodiments of the present disclosure.

FIG. 8B illustrates an optical image of an example single biologicalcell held and aspirated by assistance of an electrically activatedmicropipette and connected to a long free end SiNT of a SiNT/W probe,consistent with one or more exemplary embodiments of the presentdisclosure.

FIG. 8C illustrates an optical image of an example electricallyactivated micropipette and a long free end SiNT of a SiNT/W probe placedin the air atmosphere, consistent with one or more exemplary embodimentsof the present disclosure.

FIG. 9A is an electrical impedance (electrical sensitivity) curvemeasured in a cellular media solution for a frequency range of about 0KHz to about 100 KHz in two situations of: SiNT not connected to asingle cell (solid line) and SiNT connected to a single cell (dashedline) , consistent with one or more exemplary embodiments of the presentdisclosure.

FIG. 9B is an electrical impedance (electrical sensitivity) curvemeasured in an air atmosphere for a frequency range of about 0 KHz toabout 100 KHz, consistent with one or more exemplary embodiments of thepresent disclosure.

FIG. 10A illustrates a cell impedance versus frequency curve for anexample of an aspirated MRC-5 single cell with three different suctionamplitudes, consistent with one or more exemplary embodiments of thepresent disclosure.

FIG. 10B is a phase response versus frequency curve for an example of anaspirated MRC-5 single cell with three different suction amplitudes,consistent with one or more exemplary embodiments of the presentdisclosure.

FIG. 10C is a cell impedance versus frequency curve for an example of anaspirated QU-DB single cell with three different suction amplitudes,consistent with one or more exemplary embodiments of the presentdisclosure.

FIG. 10D is a phase response versus frequency curve for an example of anaspirated QU-DB single cell with three different suction amplitudes,consistent with one or more exemplary embodiments of the presentdisclosure.

FIG. 11A illustrates a confocal microscope image of an example of acontrol MRC-5 cell before mechanical aspiration, consistent with one ormore exemplary embodiments of the present disclosure.

FIG. 11B illustrates a confocal microscope image of an example of aMRC-5 cell after mechanical aspiration, consistent with one or moreexemplary embodiments of the present disclosure.

FIG. 11C illustrates a confocal microscope image of an example of acontrol QU-DB cell before mechanical aspiration, consistent with one ormore exemplary embodiments of the present disclosure.

FIG. 11D illustrates a confocal microscope image of an exemplary QU-DBcell after mechanical aspiration, consistent with one or more exemplaryembodiments of the present disclosure.

FIG. 12A is a cell impedance versus frequency curve for an exemplaryaspirated HT-29 single cell with three different suction amplitudes,consistent with one or more exemplary embodiments of the presentdisclosure.

FIG. 12B is a phase response versus frequency curve for an exemplaryaspirated HT-29 single cell with two different suction amplitudes,consistent with one or more exemplary embodiments of the presentdisclosure.

FIG. 12C is a cell impedance versus frequency curve for an exemplaryaspirated SW-48 single cell with three different suction amplitudes,consistent with one or more exemplary embodiments of the presentdisclosure.

FIG. 12D is a phase response versus frequency curve for an exemplaryaspirated SW-48 single cell with three different suction amplitudes,consistent with one or more exemplary embodiments of the presentdisclosure.

DETAILED DESCRIPTION

The following detailed description is presented to enable a personskilled in the art to make and use the methods and systems disclosed inexemplary embodiment of the present disclosure. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present disclosure. However, it will be apparent toone skilled in the art that these specific details are not required topractice the disclosed exemplary embodiments. Descriptions of specificexemplary embodiments are provided only as representative examples.Various modifications to the exemplary implementations will be readilyapparent to one skilled in the art, and the general principles definedherein may be applied to other implementations and applications withoutdeparting from the scope of the present disclosure. The presentdisclosure is not intended to be limited to the implementations shown,but is to be accorded the widest possible scope consistent with theprinciples and features disclosed herein.

Disclosed herein is an exemplary nanostructured electrical probe (atungsten-(W—) supported silicon nanotube-(SiNT-) based electrical probe(SiNT/W probe)) and an exemplary method for fabrication thereof. Theprobe may be considered for a non-invasive measurement of electricalresponses of a cell.

In an aspect, the present disclosure describes an exemplaryelectromechanical method to detect changes in the electrical propertiesof a single cell, between normal and cancerous states during amechanical deformation. The method is based on the role of actinmicrofilaments within a cell in modulation of the ion channel activityand consequently based on the electrical response (e.g. electricalimpedance, phase, etc.) of a cell during a mechanical deformation, suchas mechanical aspiration. In some aspects, the method may be consideredas a new label-free electromechanical cancer diagnosis and cancerprogression monitoring method with a single-cell resolution.

FIG. 1 illustrates a schematic of one example of a SiNT/W probe 100,consistent with one or more exemplary embodiments of the presentdisclosure, which may be configured for measuring an electrical responsefrom a biological cell. Referring to the implementation shown in FIG. 1,the SiNT/W probe 100 may include a tungsten (W) microwire 101 with asharpened tip section 102 that may be coated with a catalyst bilayer anda plurality of silicon nanotube (SiNTs) electrodes 103 verticallyaligned on the catalyst bilayer. The SiNTs 103 may be configured toconnect or attach to a biological cell and measure an electricalresponse of the biological cell that is in contact with the electrodes.Accordingly, a long free-end SiNT 104 may be used among other SiNTswithin the array. The long free-end SiNT 104 may be configured forconnecting to a biological cell and penetrating the biological cell forfurther electrical measurement purposes according to one or more aspectsof the present disclosure.

As used herein, a microwire may be a fine wire with a circularcross-section and a diameter less than 2000 μm. The microwire 101 maybe, for example a tungsten (W) microwire. In certain examples, thetungsten (W) microwire 101 may have a diameter less than about 500 μm. Asharpened tip of the sharpened tip section 102 of the tungsten (W)microwire where the long free-end SiNT 104 may be connected may have adiameter of about 200 nm.

In some exemplary implementations, the catalyst layer may include acatalyst bilayer. The catalyst bilayer, as used herein, is defined as adouble-layered catalyst with one layer of a first catalyst coated onanother layer of a second catalyst. The catalyst bilayer may include alayer of Nickel (Ni) with a thickness of, for example about 10 nm toabout 40 nm over a layer of gold (Au) with a thickness of, for exampleabout 1 nm to about 4 nm forming a catalyst bilayer (Ni—Au).

In an implementation, the array of nanotubes 103 may include a pluralityof vertically-aligned silicon nanotubes (SiNTs) that may be grown on thecatalyst bilayer. The SiNTs may have a thickness or diameter of, forexample less than about 100 nm.

FIG. 2 illustrates an example of a method 200 for fabricating the SiNT/Wprobe 100, consistent with one or more exemplary embodiments of thepresent disclosure. The method 200 may include the steps of: first,sharpening one end of a tungsten (W) microwire to form a tungsten (W)needle having a sharp pointed tip (step 201), second, cleaning thetungsten (W) needle to obtain a cleaned tungsten (W) needle (step 202),third, forming a catalyst bilayer on the sharpened tip of the cleanedtungsten (W) needle (step 203), fourth, growing a plurality of siliconnanotubes (SiNTs) on the catalyst bilayer to form a SiNT/W needle (step204), fifth, doping the SiNT/W needle using a doping furnace to form adoped conductive SiNT/W needle (step 205),and sixth, coating a goldlayer on top of the SiNTs of the doped conductive SiNT/W needle (step206).

Referring to the first step 201, an initially supplied tungsten (W)microwire may be sharpened from one end, for example via anelectrochemical etching process to form a tungsten (W) needle having asharp pointed tip. FIG. 3 shows four schematics of one example of theinitially supplied tungsten (W) microwire 301, the tungsten (W) needle302, and the cleaned tungsten (W) needle having a catalyst bilayer onthe sharp tip 303 and the SiNT/W needle 304 during the fabricationprocess, consistent with one or more exemplary embodiments of thepresent disclosure. Referring to FIG. 3, a schematic of one example of atungsten (W) microwire 301 and the obtained tungsten (W) needle 302after step 201 is illustrated.

Moving on to the second step 202, cleaning the tungsten needle may becarried out with immersing the tungsten needle in a cleaning solution,for example, a solution of acetone and buffer HF.

Moving on to the third step 203, the catalyst bilayer may be formed viaa two-step deposition process, using, for example, an electron beamcoating system via placing the needle in a position in which the top ofthe needle can be located in front of a target plume. The formation ofthe catalyst bilayer may include two steps of: first, holding thecleaned tungsten needle under a gold plume to coat a layer of gold onthe sharp tip to form a first catalyst layer; and second, holding thecleaned tungsten needle having the first catalyst layer under a Nickelplume to coat a layer of nickel over the first catalyst layer to yield acatalyst bilayer (Ni—Au). Accordingly, a thin layer of gold, with athickness ranging, for example from about 1 nm to about 4 nm may becoated on top of the sharp tip. Subsequently, a layer of nickel with athickness ranging, for example from about 10 nm to about 40 nm may becoated over the gold layer. Referring to FIG. 3, a schematic of atungsten needle 303 having a catalyst bilayer formed over its sharpenedtip is illustrated.

Moving on to the fourth step 204, the SiNTs may be grown via avapor-solid-liquid (VLS) process using a low-pressure chemical vapordeposition (LPCVD) chamber. The VLS process may be carried out by theassistance of, for example H2 and SiH4 gases at a temperature in a rangeof about 400° C. to about 600° C. and at a pressure of about 1 mTorr. Anexample of the obtained SiNT/W needle 304 from step 204 is schematicallyillustrated in FIG. 3.

Moving on to the fifth step 205, the doping step may be carried out byan element of group five of the periodic table, for example,phosphorous. In an implementation, the doping step may be carried out ina phosphorous doping furnace. The SiNT/W needle may be held in thephosphorous doping furnace at a temperature of, for example about 700°C. for about 10 minutes.

Moving on to the final step 206, the gold layer may be coated over theSiNTs via a sputtering system. The thickness of the gold layer may be,for example about 5 nm.

In another aspect, a single-cell-based electromechanical method forcancerous state detection of a single biological cell is described. Thebiological cell may be a biological cell having an elastic cell membranewith a defined membrane elasticity, for example, epithelial,endothelial, or mesenchymal cells. This method may be used, for example,for cancer diagnosis, detecting cancer transformation or progression,detecting cancer cells among biological cells, investigating metastaticstage, or generally for cancerous state determination of a malignanttissue.

FIG. 4 shows an example of a method 400 for detecting the cancerousstate of a single biological cell, consistent with one or more exemplaryembodiments of the present disclosure. The method 400 may include stepsof first, preparing a suspension of individually suspended biologicalcells (step 401), second, extracting a single cell from the suspension(step 402), third, holding the extracted single cell from the suspension(step 403), fourth, measuring a first electrical response of the heldsingle cell (step 404), fifth, step-wised mechanical aspirating the heldsingle cell to form a mechanically deformed cell (step 405), and sixth,measuring an electrical response of the held single cell after each stepof the mechanical aspirating (step 406).

In step 401, a suspension of biological cells including individual cellsthat are distributed within the suspension may be prepared via a processwith steps of, culturing a plurality of biological cells onto asubstrate, washing the cultured cells, trypsinizing the cultured cellsto detach the cultured cells from the substrate and form a solution, andcentrifuging the solution to separate a cell suspension that includesindividually suspended biological cells. Accordingly, a plurality ofbiological cells may be cultured onto a substrate, for example a glasssubstrate. The cells may be cultured in a culture medium, for example, aRoswell Park Memorial Institute-1640 (RPMI-1640) medium. The culturemedium may be supplemented with a serum-supplement, for example, Fetalbovine serum (FBS) including Fetal bovine with an amount of about 5% andthe culture medium may be further supplemented with an antibiotic, forexample, penicillin/streptomycin with an amount of about 1%. Then, thecultured cell may be washed with a buffer solution, for example, aPhosphate-buffered saline (PBS) solution to remove the remained culturedmedia and supplements from the cultured cells. Subsequently, thecultured and washed cells may be trypsinized by assistance of adding asolution including trypsin and EDTA to the cultured cells in order todetach the cultured cells from the substrate and form a solutionincluding the cells. Finally, the obtained solution including thecultured cells may be centrifuged to discard the trypsinizing solutionand separate a cell suspension including individually suspendedbiological cells.

Referring to second step 402 and subsequently, third step 403, a singlecell may be extracted from the suspension and held for a while byassistance of an electrically activated micropipette. The electricallyactivated micropipette may be a glass micropipette with a diameter in arange of about 4.5 μm to about 5 μm, which may be coated with anelectrically conductive layer, for example, a gold (Au) layer. The gold(Au) layer may be coated with a thickness of, for example about 10 nmover the glass micropipette via, for example a sputtering system.

Moving on to step 404, an electrical response of the held cell, forexample, an electrical impedance of the cell membrane of the held cellmay be measured using an electrical probe connected to the cell. Theelectrical probe may include a SiNT/W probe, designed and fabricatedpursuant to the teachings of the present disclosure.

Moving on to step 405, the held single cell may be aspirated byassistance of the same electrically activated micropipette, which wasused before for extracting step 402 and holding step 403. Theelectrically-activated micropipette may be assembled on a microinjectionmicroscope to supply the electrically activated micropipettedisplacements needed in steps 402 and 403. In addition, the negative andpositive pressure for aspirating the single cell may be applied to theglass micropipette by assistance of a movable water reservoir of themicroinjection microscope. Displacing the water reservoir up or downleads to a suitable pressure to pull in or force away the cell.Furthermore, a micromanipulator may be utilized to adjust eachmicropipette position. Also, the aspirated leading edge of the cellsurface may be monitored using an inverted microscope equipped with adigital camera assembled on the microinjection microscope.

Moving on to step 406, an electrical response of the held single cellafter each step of mechanical aspirating of step 405 may be measured.Then, the cancerous state of the single cell may be determined based onthe changes of the electrical response measured in step 404 and theelectrical responses measured in step 406. Since, the electricalproperties of a normal or healthy cell, for example electrical impedanceof the cell membrane may be affected significantly by the mechanicalproperties of the cell, sharp and significant changes in the measuredelectrical responses from the single cell after cell mechanicaldeformation may indicate that the extracted single cell from thesuspension is a healthy cell. While, no or small alterations inelectrical responses measured during mechanical deformation may indicatethat the selected and processed single cell via the method 400, pursuantto the teachings of the present disclosure is a cancer cell.

FIG. 5A shows a schematic of an example of a single biological cell 501,which is extracted from a suspension including individually suspendedcells, consistent with one or more exemplary embodiments of the presentdisclosure. Cell 501 is held and aspirated by assistance of anelectrically activated micropipette 502 and it is in contact with asilicone nanotube (SiNT) 503 of a SiNT/W probe similar to a siliconenanotube (SiNT) 104 described hereinabove. SiNT 503 is a long free-endSiNT among the SiNTs array that is formed over the sharpened tip of theprobe.

It should be understood that the structure changes in actinmicrofilament network of a cell due to a mechanical force may be acriterion for diagnosis between cancerous and healthy cells as well asbetween benign and metastatic cells. For example, a mechanicalaspiration mechanism applied on a healthy or benign cell may cause asignificant alteration in actin microfilament configuration andsubsequently a significant change in an electrical response of the cell,for example electrical impedance or phase. While, a similar mechanicalaspiration may not cause any observable change in such electricalresponses in case of a cancerous or metastatic cell.

Accordingly, FIG. 5B shows a schematic of the actin microfilamentconfiguration and distribution for a non-aspirated cell illustrating theelectrically activated micropipette 502 near the single cell 501 asdescribed hereinabove and a schematically actin microfilamentdistribution 504 of the cell, consistent with one or more exemplaryembodiments of the present disclosure. Correspondingly, FIG. 5C shows asimilar schematic actin microfilament configuration and distribution foran aspirated cell, consistent with one or more exemplary embodiments ofthe present disclosure.

In another aspect, an electromechanical system for detecting cancerousstate of a single cell is described. The system may include a firstaspirating mechanism to extract and hold a single cell and apply amechanical aspiration to the single cell; and a second electricalmeasurement mechanism to measure an electrical response of the singlecell. The cancerous state of the single cell may be detected based onthe changes of the measured electrical responses.

FIG. 6 illustrates an example of an electromechanical system 600,configured to detect cancerous state of a single cell, consistent withone or more exemplary embodiments of the present disclosure. Theelectromechanical system 600 may be utilized to implement the method 400of FIG. 4 for detecting cancerous state of a single cell as describedabove. Exemplary system 600 includes an aspirating mechanism 601configured to extract and hold a single cell and then applying amechanical aspiration to the single cell and an electrical measurementmechanism 602 configured to measure an electrical response of the singlecell. In an implementation, the aspirating mechanism 601 may include anelectrically activated glass micropipette (similar to electricallyactivated micropipette 502) with two ends, which is coated with a goldlayer. The electrically activated glass micropipette may be assembled ona microinjection microscope from one end, while having a nozzle at theother end to apply and transfer the mechanical aspiration to the singlecell.

Referring to FIG. 6, the electrical measurement mechanism 602 mayinclude: an electrical probe 603 configured to be connected to the heldcell by the electrically activated glass micropipette for electricalmeasurements; a signal controlling system 604 configured for applying anelectrical signal to the extracted and held single cell connected to theelectrical probe 603 and acquiring the corresponding electrical responseof the extracted and held single cell connected to the electrical probe603, and a data processor 605 configured for recording and analyzing theelectrical response in order to detect the cancerous state of the singlecell. The electrical probe 603 may include a tungsten-supported siliconenanotube-based (SiNT/W) probe assembled on a microinjection microscopeopposite to the electrically activated glass micropipette 502 to connectand penetrate a long free-end SiNT 503 to the single cell held by theelectrically activated glass micropipette.

With further reference to FIG. 6, the signal controlling system 604 mayinclude an AC signal source 606 configured for applying the electricalsignal to the electrical probe 603 and a data acquisition module 607configured for acquiring the electrical response corresponding to theelectrical signal from the electrical probe 603. The AC signal source606 may be configured to apply a voltage of, for example, about 40 mV tothe electrical probe 603. Accordingly, the applied voltage may cause afrequency that ranges from about 100 Hz to about 100 KHz.

EXAMPLES Example 1 Fabricating a SiNT/W Probe

In this exemplary scenario, a tungsten (W) needle as a support for aSiNT/W probe may be made from an initial W microwire with a diameter ofabout 500 μm, using an electrochemical etching process. An optical imageof an example sharpened tip of a W needle is shown in FIG. 7A,representing the formed tip at one end of the needle with a diameter ofabout 200 nm. The W needle may be washed and cleaned with a solution ofacetone and Buffer HF. Subsequently, the cleaned needle may be held inan electron beam coating system) to deposit a bilayer catalyst ofNickel-Gold (Ni—Au) on the sharp tip of the cleaned W needle. During thedeposition process, the needle may be placed in a position in which, thetop portion of the needle is located in front of the target plume. Thedeposition may begin at a base pressure of about 10⁻⁶ Ton. A thin layerof gold, with a thickness of about 2 nm may be coated on the tip of theprobe. Subsequently, another layer of nickel with a thickness of about20 nm may be coated over the gold layer. In a next step, the growth ofSiNTs on the catalyst bilayer may be achieved via placing the W needlecoated with the Ni—Au catalyst over the tip in a LPCVD chamber (SenslranCo. Iran). The SiNTs may be grown over the catalyst bilayer by theassistance of H₂ and SiH₄ gases at a base pressure of about 1 mTorr andat a temperature of about 450° C. to form the SiNT/W needle. A magnifiedzone of FIG. 7A represented by 701 is shown in FIG. 7B. This figureillustrates a field emission scanning electron microscope (FESEM) imageof an example grown array of SiNTs over the sharpened tip of a W needle.Then, the SiNT/W needle may be transferred into a phosphorous dopingfurnace and held at a temperature of about 700 ° C. for about 10 minutesto enhance the conductivity of the nanotubes by the diffusion ofphosphorous dopants atoms. Finally, a gold layer with a thickness ofabout 5 nm may be coated on top of the nanotubes with the assistance ofa sputtering system to form the SiNT/W probe. FIG. 7C illustrates afield emission scanning electron microscope (FESEM) micrograph of anexample of a single silicone long free end nanotube (SiNT) 702 of FIG.7B among the array of SiNTs over the sharpened tip of a W needle, themost appropriate SiNT among the SiNTs array for further superficial andnegligibly invasive cell connection and penetration electricalmeasurement purposes causing the least electrical noises. This figurealso shows a diameter of about 70 nm for the formed SiNT on the probetip.

Example 2 Investigation of the Electrical Sensitivity

In this example, the basic electrical sensitivity of the disclosedsystem may be characterized by entering and placing a SiNT/W probe andan electrically activated glass micropipette of the system pursuant tothe present disclosure in an ionic cellular media solution containing abiological cell suspension, followed by comparing the sensitivity beforeand after connecting the SiNT/W probe to the cell grasped by themicropipette, and finally by scaling under a dry atmosphere.

FIGS. 8A-8C show optical images of three situations configured formeasurement of the electrical response before (FIG. 8A) and after (FIG.8B) the connection of the SiNT/W probe to the cell within a cellularmedia solution and finally in a dry ambient atmosphere (FIG. 8C),consistent with one or more exemplary embodiments of the presentdisclosure.

Referring to FIG. 8A, an optical image of a long free end SiNT 801 of aSiNT/W probe and an electrically activated micropipette 802 placed in acellular media solution 803 is illustrated. A single biological cell 804may be selected and held within the cellular media solution 803 byassistance of the electrically activated micropipette 802, while theprobe may be placed over the cell without any connections to the cell.FIG. 8B shows a second situation similar to FIG. 8A, while the longfree-end SiNT 801 connected and penetrated the single biological cell804 aspirated by the electrically activated micropipette 802.Subsequently, FIG. 8C shows a third exemplary situation, in which thelong free-end SiNT 801 of a SiNT/W probe and the electrically activatedmicropipette 802 are placed in a dry air atmosphere 805.

FIGS. 9A and 9B show corresponding electrical impedance (electricalsensitivity) values that are measured in a frequency range of about 0KHz to about 100 KHz for the three exemplary situations described aboveand shown in FIGS. 8A-8C, consistent with one or more exemplaryembodiments of the present disclosure. Referring to FIG. 9A,representing the impedance values for the first and second situationswithin the cellular media solution, the impedance curves are obtainedfor the cases, where the SiNT is not connected to the single cell(designated by the solid line) and where the SiNT is connected to thesingle cell (designated by the dashed line) are within a similar rangeof about 1 kΩ to about 7 kΩ, while regarding FIG. 9B, the impedancemagnitudes increased significantly to a range of about 0 kΩ to about 140kΩ when the ambient was changed from a cellular media solution to an airatmosphere in a fixed distance between the SiNT and the micropipette(about 7 μm).

Example 3 Detecting the Cancerous State of a Single Cell

In this example, the electromechanical method and system may be used todetect the cancerous state of a single cell. To this end, healthy lungcells (MRC-5) and cancerous lung cells (QU-DB) were utilized. The MRC-5was derived from a healthy or normal lung tissue and QU-DB was derivedfrom a human lung carcinoma tissue. For cell culturing, cells weremaintained in a CO₂ incubator (37° C., 5% CO₂, 95% air) in a RPMI-1640medium supplemented with 5% fetal bovine serum (Gibco), and 1%penicillin/streptomycin (Gibco). The fresh medium was replaced everyday. Prior to each experiment, cells were trypsinized in order to bedetached from the substrate and were suspended in the culture medium. Tominimize the effect of trypsinization, the procedure was carried out inless than 4 minutes at a temperature of about 20-22° C. Single cellssuspended within the prepared suspension were extracted, held andaspirated by an electrically activated glass micropipette having anozzle with an inner diameter of about 5 μm. Then, the SiNT/W probe wasconnected to the aspirated cell and an electrical response (impedancemagnitude and phase) were measured for different suction forces appliedby the micropipette.

FIGS. 10A-10D show the results of the electrical measurements fromaspirated MRC-5 and QU-DB cells with various suction forces, consistentwith one or more exemplary embodiments of the present disclosure. Therepresentative recorded data in FIGS. 10A and 10B show a clear increasein the cell impedance and phase with increasing mechanical stretchamplitudes from F1 to F3 in healthy cells (MRC-5), whereas no noticeableimpedance and phase changes were observed in malignant cells afterincreasing the aspiration with the same suction forces as shown in FIGS.10C and 10D.

The suction forces during cell aspiration resulted in different lengthsof the cell that flowed into the pipette (Lp) due to the mechanicalproperties of each individual cell. The Lp value was determined frommicroscopy images. Table 1 shows the Lp (μm) values and correspondingchanges in electrical responses (electrical impedance (kΩ) and phase(θ)) for each single cell that was aspirated by three increasing varioussuction forces of F1, F2 and F3. The data shows that changes in theelectrical parameters initiated from mechanical aspiration in healthylung cells (MRC-5) were about 10 times higher than those of aspiratedcancerous cells (QU-DB). These results suggest that bioelectricalproperties of a healthy cell have a strong correlation with itsmechanical function.

Furthermore, the effects of cancerous transformation and cell aspirationon actin microfilament distribution on control and stretched MRC-5 andQU-DB cell samples were assessed by inverted confocal microscopy. Priorto imaging, cells were fixed in about 4% formaldehyde for about 15 minand permeabilized in PBS (with the concentration of about 1%) for about5 min to 10 min at room temperature. Then, all samples were washed andstained with the phalloidin-FITC conjugate (Green)) and incubated forabout 30 min to 45 min. The cell nuclei were stained with propidiumiodide (PI). The Leica Application Suite Advanced Fluorescence (LAS AF)software (Leica Microsystems) was utilized to analyze the confocalmicroscopy images.

FIG. 11A shows a confocal microscope image of a control MRC-5 cellbefore aspiration, while a confocal microscope image of an aspiratedMRC-5 cell is shown in FIG. 11B. FIGS. 11A and 11B show that themechanical aspiration results in major alterations in the actinmicrofilaments for MRC-5 cell. Also, corresponding images are shown inFIGS. 11C and 11D representing confocal microscopy images of a controlQU-DB cell before aspiration (FIG. 11C) and after aspiration (FIG. 11D)illustrating less alterations in cell actin microfilaments structure fora QU-DB in comparison with those alterations for MRC-5 cell. It may beconcluded that cancerous transformation resulted in the rebundling ofactin microfilaments during aspiration mechanism, so the mechanicalproperties and subsequently electrical properties of a cancerous cellwould remain the same before and after cell aspirating. As a result,confocal microscopy showed the crucial role of actin microfilaments incells that had highly reduced electromechanical behavior aftermetastatic progression. It showed the distinct differences in actinmicrofilament configurations between the control samples of healthy andmalignant lung cells. The images showed that the actin microfilamentsare bundled and remodeled in QU-DB cells during mechanical aspiration,while the actin microfilaments configurations of a healthy MRC-5 cell issignificantly changed applying a mechanical aspiration.

Example 4 Detecting Cell Metastasis Progression

In this example, in order to elucidate the effect of metastasisprogression of cancer cells on their electromechanical response, someexperiments were performed on colon primary (HT-29) and progressive(SW-48) malignant cells. The colon primary or benign cells (HT-29) andcolon progressive or metastatic (SW-48) malignant cells were obtainedfrom the National Cell Bank of Iran, Pasteur Institute. Both types ofcells were cultured, suspended and their electrical properties weremeasured before and after mechanical aspiration, identical to themethods and techniques described in connection with example 3.

FIGS. 12A-12D show the results of the electrical measurements fromaspirated HT-29 and SW-48 cells after applying three various suctionforces (F1, F2 and F3) during mechanical aspiration, consistent withexemplary embodiments of the present disclosure. The representativerecorded data in FIGS. 12A and 12B show an increase in the cellimpedance and phase with increasing mechanical stretch amplitudes fromF1 to F3 for HT-29, whereas no noticeable impedance and phase changeswere observed for SW-48 after increasing the aspiration with the samesuction forces as shown in FIGS. 12C and 12D.

The suction forces during cell aspiration resulted in different lengthsof the cell that flowed into the pipette (Lp) due to the mechanicalproperties of each individual cell. The Lp value was determined frommicroscopy images. Table 1 shows the Lp (μm) values and correspondingchanges in electrical responses (electrical impedance (kΩ) and phase(θ)) for each single cell that was aspirated by three increasing varioussuction forces of F1, F2 and F3. These data shows that the averageimpedance and phase variation of an aspirated HT-29 cell wereapproximately 2-fold higher than those of a SW-48 cell.

TABLE 1 Change in cell electrical parameters due to the mehanicalaspiration

What is claimed is: 1- An electrical probe for measuring an electricalresponse from a biological cell, comprising: a microwire having asharpened tip section, a catalyst layer formed on the sharpened tipsection of the microwire; and an array of nanotube electrodes verticallyaligned on the catalyst layer, wherein, the array of nanotube electrodesare configured to measure the electrical response of the biological cellin contact with the electrodes. 2- The electrical probe according toclaim 1, wherein one of the nanotube electrodes is located on a tip ofthe sharpened tip section and is longer than remaining nanotubeelectrodes of the nanotube electrodes. 3- The electrical probe accordingto claim 1, wherein the microwire includes a tungsten microwire. 4- Theelectrical probe according to claim 2, wherein the microwire has adiameter less than 2000 μm. 5- The electrical probe according to claim1, wherein a tip of the sharpened tip section of the microwire has adiameter about 200 nm. 6- The electrical probe according to claim 1,wherein the catalyst layer includes a bilayer catalyst having a Nickellayer (Ni) and a Gold layer (Au). 7- The electrical probe according toclaim 6, wherein the Gold layer (Au) has a thickness in a range of 1 nmto 4 nm and the Nickel layer (Ni) has a thickness in a range of 10 nm to40 nm. 8- The electrical probe according to claim 1, wherein thenanotube electrodes include a plurality of silicone nanotubes (SiNTs).9- A method for fabricating a tungsten (W) supported silicon nanotube(SiNT) based electrical probe (SiNT/W probe) comprising steps of:sharpening one end of a tungsten (W) microwire to form a tungsten (W)needle having a sharpened tip section; forming a catalyst bilayer on thesharpened tip section of the tungsten (W) needle; growing a plurality ofsilicon nanotubes (SiNTs) on the catalyst bilayer to form a SiNT/Wneedle; doping the SiNT/W needle using a doping furnace to form a dopedconductive SiNT/W needle; and coating a gold layer on top of the SiNTsof the doped conductive SiNT/W needle to form a SiNT/W probe, whereinthe electrical probe is configured to measure an electrical response ofa biological cell contacting the silicon nanotubes (SiNTs). 10- Themethod according to claim 9, wherein a silicon nanotube located on a tipof the sharpened tip section is longer than remaining silicon nanotubesof the plurality of silicon nanotubes (SiNTs). 11- The method accordingto claim 9, wherein sharpening the one end of a tungsten (W) microwireis done via an electrochemical etching process. 12- The method accordingto claim 9, wherein the tungsten (W) needle is cleaned via immersion ina solution including acetone and buffer HF. 13- The method according toclaim 9, wherein forming a catalyst bilayer on the sharpened tip sectionof the tungsten (W) needle includes a two-step deposition process usingan electron beam coating system, the process comprising: holding thetungsten (W) needle under a gold plume to coat a layer of gold on thesharp tip to form a first catalyst layer; and holding the tungsten (W)needle having the first catalyst layer under a Nickel plume to coat alayer of Nickel over the first catalyst layer to form the catalystbilayer (Ni-Au) on the sharp tip of the tungsten (W) needle. 14- Themethod according to claim 9, wherein growing the plurality of SiNTsincludes growing the plurality of SiNTs via a vapor-solid-liquid (VLS)process using a Low-Pressure Chemical Vapor Deposition (LPCVD) system.15- The method according to claim 13, wherein: the VLS process is doneusing H₂ and SiH₄ gases; and the VLS process is done at a temperaturerange of about 400° C. to 600° C. 16- The method according to claim 13,wherein the VLS process is done at a pressure of about 1 mTorr. 17- Themethod according to claim 9, wherein the doping furnace includes aphosphorous doping furnace. 18- The method according to claim 17,wherein the SiNT/W needle is held in the phosphorous doping furnace at atemperature of about 700° C. for about 10 minutes. 19- The methodaccording to claim 9, wherein coating a gold layer on top of the SiNTsof the doped conductive SiNT/W needle is done using a sputtering system.20- The method according to claim 20, wherein the gold layer has athickness of 5 nm.